Dihydrofolate reductase (DHFR) will be purified from S. faecium (isoenzyme I), rat liver (all isoenzymes) and human cells. The structure of the DHFR from these sources will be studied by sequencing, chemical modification, and NMR after specific labeling with 13C. Spin-labeled antifolate analogues will be further purified and used to determine internal motion of such ligands when bound to DHFR and to determine distance between the unpaired spin of the bound ligand and 13C nuclei in specific amino acid side-chains. Sequence data will be analyzed for information indicating similarity of the overall structure of different DHFRs, and the primary sequence differences that are the molecular bases of differences in function. NMR and other data for various complexes will be examined for indications of differences in the modes of binding of different antifolates that can be correlated with inhibition constants and specificity. Data will also be examined for indications of the catalytic mechanisms.